Our goal is to understand the molecular mechanisms responsible for the initiation and prevention of calcification in the artery media. Our working hypothesis is that blood contains a causative agent(s) for medial artery calcification, and that the ability of this serum nucleator to initiate calcification in the elastic lamellae of the artery media is normally prevented by the opposing action of calcification inhibitors such as the vitamin K-dependent matrix Gla protein (MGP), an inhibitor which is secreted by smooth muscle cells adjacent to the elastic lamellae in the artery media. This blood borne theory of medial artery calcification predicts that calcification can occur in vivo whenever the initiation of medial calcification driven by the serum nucleator exceeds the capacity ofinhibitors such as MGP to prevent this calcification. Our initial study shows that serum contains a potent nucleator of elastin calcification, and that this agent is a 55-150kDa protein. Our first aim is to understand the serum nucleator of medial artery calcification, and we have designed experiments to identify the nucleator, to use recombinant nucleator in a variety of studies of nucleator structure-function, and to test the hypothesis that serum nucleator activity drives artery calcification in vivo. Our second aim is to understand the factors that determine the extent of medial calcification in the rat, and we have designed experiments to test the ability of bone resorption inhibitors to prevent medial calcification in uremia, and to arrest the progression of medial calcification once it has begun. We will also determine whether warfarin acts synergistically with uremia to accelerate medial calcification. Our third aim is to identify the molecular mechanisms by which medial artery calcification is normally prevented by matrix Gla protein. Pilot studies show that MGP is highly expressed by the artery in organ culture, that warfarin causes medial calcification in organ culture, and that addition of MGP to medium prevents this calcification. We have designed experiments using artery organ culture to determine the mechanisms by which MGP normally inhibits medial artery calcification; the relation between MGP structure and its activity as a calcification inhibitor; and the factors, which regulate MGP expression and phosphorylation by vascular cells.